Cytoplasmic actin in neuronal processes as a possible mediator of synaptic plasticity

We have demonstrated that, after permeation with saponin and decoration with S-1 myosin subfragment, the cytoplasmic actin is organized in filaments in dendritic spines, dendrites, and axon terminals of the dentate molecular layer. The filaments are associated with the plasma membrane and the postsynaptic density with their barbed ends and also in parallel with periodical cross bridges. In the spine stalks and dendrites, the actin filaments are organized in long strands. Given the contractile properties of actin, these results suggest that the cytoplasmic actin may be involved in various forms of experimentally induced synaptic plasticity by changing the shape or volume of the pre- and postsynaptic side and by retracting and sprouting synapses.     

Cytoplasmic actin patterns in Physarum as revealed by NBD-phallacidin staining

Fluorescently labeled phallacidin, a F-actin specific drug, was used to demonstrate the morphological variety in the cytoskeletal actin pattern of thin-spread plasmodia of the acellular slime mould Physarum polycephalum. The patterns observed in phallacidin-stained specimens consisted of a polygonal network in the anterior region, and of longitudinal as well as helically twisted fibrils in plasmodial strands of the posterior region. These observations are in complete accordance with our recent results obtained on comparable plasmodia by immunofluorescence microscopy using specific antibodies against actin.     

Gelsolin as a calcium-regulated actin filament-capping protein.

Various concentrations of gelsolin (25-100 nM) were added to 2 microM polymerized actin. The concentrations of free calcium were adjusted to 0.05-1.5 microM by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nM) that exceed the concentration of filament ends ( approximately 2 nM), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium-regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity.     

Cytoplasmic actin and cochlear outer hair cell motility

Summary Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaing cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.     

Cytoplasmic dynein is a vesicle protein.

Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.     

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.     

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).     

Lasp-2 expression, localization, and ligand interactions: a new Z-disc scaffolding protein

The nebulin family of actin-binding proteins plays an important role in actin filament dynamics in a variety of cells including striated muscle. We report here the identification of a new striated muscle Z-disc associated protein: lasp-2 (LIM and SH3 domain protein-2). Lasp-2 is the most recently identified member of the nebulin family. To evaluate the role of lasp-2 in striated muscle, lasp-2 gene expression and localization were studied in chick and mouse tissue, as well as in primary cultures of chick cardiac and skeletal myocytes. Lasp-2 mRNA was detected as early as chick embryonic stage 25 and lasp-2 protein was associated with developing premyofibril structures, Z-discs of mature myofibrils, focal adhesions, and intercalated discs of cultured cardiomyocytes. Expression of GFP-tagged lasp-2 deletion constructs showed that the C-terminal region of lasp-2 is important for its localization in striated muscle cells. Lasp-2 organizes actin filaments into bundles and interacts directly with the Z-disc protein alpha-actinin. These results are consistent with a function of lasp-2 as a scaffolding and actin filament organizing protein within striated muscle Z-discs.     

Cytoplasmic 8 S glucocorticoid receptor binds to actin filaments through the 90-kDa heat shock protein moiety

The glucocorticoid receptor exists in the cytoplasm of hormone-untreated cells as a complex with the 90-kDa heat shock protein (HSP90). Glucocorticoids induce dissociation of the glucocorticoid binding protein from HSP90 and translocation of the receptor to the nucleus. HSP90 binds to actin filaments, and calmodulin or tropomyosin inhibits the binding. We present here evidence that the HSP90-containing glucocorticoid receptor complexes (8 S receptor) bind to filamentous actin in vitro while the HSP90-free form of the receptor does not. The binding was detectable for both the crude cytosolic fractions and the partially purified 8 S glucocorticoid receptor. Purified HSP90 or tropomyosin completely abolished the binding. Calmodulin also inhibited the binding in a Ca(2+)-dependent manner. From these results, we conclude that the glucocorticoid receptor complex is able to bind actin filaments via the HSP90 moiety. The binding may provide an anchoring mechanism for the glucocorticoid receptor in the cytoplasm.     

Cytoplasmic actin A3 gene promoter injected as supercoiled plasmid is transiently active inBombyx mori embryonic vitellophages

Summary Freshly deposited eggs ofBombyx mori were microinjected with supercoiled plasmid DNA which carried the -galactosidase coding sequence ofEscherichia coli inserted in place of the coding sequence of theB. mori cytoplasmic actin A3 gene. Transient expression of this fusion gene in the embryo was determined by in situ histochemical detection of enzyme activity. After injection of the plasmid at different stages of embryonic development, -galactosidase activity depending on the injected DNA was only detected in the vitellophages. This indicates the presence of active transactivators of the actin A3 gene promoter in this cell type. Tissue specificity of the fusion gene expression could be related to the early polyploidization of vitellophages, a process which would favour the stability of the nuclear pool of injected plasmids. The activity of the transgene in vitellophages was detectable at 24–33 h of egg development, the stage presumed for the onset of zygotic gene expression, up to the end of embryogenesis. This gene transfer system is thus promising to analyse thecis regulatory sequences of the actin A3 gene and could be utilized for other ubiquitous genes. Correspondence to: M. Coulon-Bublex     

Spectrin and protein 4.1 as an actin filament capping complex

Spectrin and protein 4.1, when added to G- or F-actin, cause the formation of short filaments, as judged by the appearance of powerful nucleating activity for G-actin polymerisation. F-Actin filaments are rapidly fragmented under physiological solvent conditions. The effect of cytochalasin E on the polymerisation reaction and the extent of reduction in the critical monomer concentration of actin when spectrin and 4.1 are added suggest that these proteins form a capping system for the more slowly growing, or 'pointed' ends of actin filaments. The interaction is not affected by calcium or by 4.9, the remaining constituent of the purified red cell membrane cytoskeleton.     

The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells

Changes in cell architecture, essentially linked to profound cytoskeleton rearrangements, are common features accompanying cell transformation. Supporting the involvement of the microfilament network in tumor cell behavior, several actin-binding proteins, including zyxin, a potential regulator of actin polymerization, may play a role in oncogenesis. In this work, we investigate the status of zyxin in Ewing tumors, a family of pediatric malignancies of bone and soft tissues, which are mainly associated with a t(11;22) chromosomal translocation encoding the EWS-FLI1 oncoprotein. We observe that EWS-FLI1-transformed murine fibroblasts, as well as human Ewing tumor-derived SK-N-MC cells, exhibit a complete disruption of their actin cytoskeleton, retaining very few stress fibers, focal adhesions and cell-to-cell contacts. We show that within these cells, zyxin is expressed at very low levels and remains diffusely distributed throughout the cytoplasm, instead of concentrating in actin-rich dynamic structures. We demonstrate that zyxin gene transfer into EWS-FLI1-transformed fibroblasts elicits reconstitution of zyxin-rich focal adhesions and intercellular junctions, dramatic reorganization of the actin cytoskeleton, decreased cell motility, inhibition of anchorage-independent growth and impairment of tumor formation in athymic mice. We observe similar phenotypic changes after zyxin gene transfer in SK-N-MC cells, suggesting that zyxin has tumor suppressor activity in Ewing tumor cells.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

New protein clustering of breast cancer tissue proteomics using actin content as a cellularity indicator

In the present study, we report the comparative proteome profiles of proteins solubilized from 37 breast cancer surgical tissues, normalized for the actin content. Blood-derived proteins were excluded from the analysis. Among the tumor-derived protein spots, a large proportion (39%) was found present in all patients. These included several glycolytic enzymes, detox and heat shock proteins, members of annexin and S100 protein families, cathepsin D, and two "rare" proteins, DDAH2 involved in the angiogenesis control, and the oncogene PARK7. Other proteins, such as psoriasin, galectin1, cofilin, peroredoxins, SH3L1, and others, showed sporadic presence and high expression level, which suggests their possible role for patient stratification.     

The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.     

Nebulin, a helical actin binding protein.

Nebulin, a giant protein (molecular mass 800 kDa) specific for the skeletal muscle of vertebrates, has been suggested to be involved in the length regulation of the thin filament as a 'molecular ruler'. Despite its size, nebulin appears to be composed mainly of small repeats of approximately 35 amino acids. We have characterized in this study the conformational and functional properties of single repeats. Complete repeats were found to bind to F-actin while a truncated one did not. One repeat is therefore the smallest unit for nebulin--actin interaction. Circular dichroism and nuclear magnetic resonance spectra measured for the peptides in water indicated a transient helical conformation. The folded region is located for them all around the conserved sequence SDxxYK. The helical conformation is strongly stabilized by anionic detergents and trifluoroethanol while uncharged or positively charged detergents have no effect. Since the surface of the actin filament is known to contain clusters of negative charges, anionic detergents may mimic the effect of an actin environment. 3D structures were calculated for three representative peptides in SDS. In vivo, the nebulin helices should form a complex with the actin filament. Based on the assumed importance of charge interactions between nebulin and actin, we propose a model for the structure of the F-actin-nebulin complex in vivo. According to that, two nebulin molecules occupy symmetrical positions along the central cleft of the actin filament bridging the two strands of the actin two-start helix. The consistency of this model with experimental data is discussed.     

Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures.

Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.     

The dynein light chain protein Tda2 functions as a dimerization engine to regulate actin capping protein during endocytosis

Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain (DLC) that works with Aim21 to regulate actin assembly during endocytosis. Here we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical DLC ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein (CP) recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a CP interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin CP recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicate the Tda2-Aim21 complex functions in actin assembly primarily through CP regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.